Allergens are known to have a direct affect on the induction of airway inflammation and asthma. The immunological mechanisms involved are now beginning to be understood. Recent evidence suggests that some allergens may be particularly potent at eliciting an immune response because the same molecule (allergen) has the ability to bind to innate receptors and serve as a conventional B and T cell antigen. Thus, Derp2 from the dust mite has been shown to behave like the mammalian protein MD-2 in recruiting bacterial lipopolysaccharide (LPS) to stimulate toll-like receptor 4 (TLR4). According to this hypothesis, the presence of Derp2 and a source of LPS (or similar lipid-based compounds), could initiate activation of the innate system, ultimately leading to stimulation of TH cells and IgE allergic reactions and associated pulmonary disease such as asthma. We propose to study the molecular details of MD-2, Derp2, and other related allergens in their binding to LPS by using a high-throughput protein expression and analysis system called yeast display. This technology will also be used for protein engineering in an effort to develop dominant negative inhibitors of MD- 2 that might serve as modulators of such adverse reactions. The specific aims are to: 1) characterize the MD-2 and Derp2 residues important in LPS and TLR4 binding, 2) analyze LPS and TLR4 binding by a panel of allergen homologs of MD-2, and 3) engineer soluble MD-2 analogs that act as dominant negative inhibitors of Derp2 in LPS recruitment. PUBLIC HEALTH RELEVANCE: For most patients with asthma, it is clear that allergies can induce the inflammatory cascade. Recent evidence suggests that some allergens may be particularly potent at eliciting an immune response because the same molecule (allergen) has the ability to bind to innate receptors and serve as a conventional B and T cell antigen. We propose to use high-throughput protein engineering and analysis to understand the molecular basis of the innate interactions, and to develop proteins that could act as specific anti-inflammatory drugs.